Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The damaged brain is largely incapable of functionally significant structural self-repair. This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, 1980; Sturrock, 1982). However, the absence of neuronal production in the adult vertebrate forebrain appears to reflect not a lack of appropriate neuronal precursors, but rather their tonic inhibition and/or lack of post-mitotic trophic and migratory support. Converging lines of evidence now support the contention that neuronal and glial precursor cells are distributed widely throughout the ventricular subependymal of the adult vertebrate forebrain, persisting across a wide range of species groups (Goldman and Nottebohm, 1983; Reynolds and Weiss, 1992; Richards et al., 1992; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995a; reviewed in Goldman, 1995; Goldman, 1997; Goldman, 1998; Goldman and Luskin, 1998; and Gage et al., 1995). Most studies have found that the principal source of these precursors is the ventricular zone (Goldman and Nottebohm, 1983; Goldman, 1990; Goldman et al., 1992; Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995), though competent neural precursors have been obtained from parenchymal sites as well (Richards et al., 1992; Palmer et al., 1995; Pincus et al., 1998). In general, adult progenitors respond to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) with proliferative expansion (Reynolds and Weiss, 1992; Kilpatrick and Bartlett, 1995; Kuhn et al., 1997), may be multipotential (Vescovi et al., 1993; Goldman et al., 1996), and persist throughout life (Goldman et al., 1996). In rodents and humans, their neuronal daughter cells can be supported by brain-derived neurotrophic factor (BDNF) (Kirschenbaum and Goldman, 1995a), and become fully functional in vitro (Kirschenbaum et al., 1994, Pincus et al., 1998a, and Pincus et al. 1998b), like their avian counterparts (Goldman and Nedergaard, 1992).
A major impediment to both the analysis of the biology of adult neural precursors, and to their use in engraftment and transplantation studies, has been their relative scarcity in adult brain tissue, and their consequent low yield when harvested by enzymatic dissociation and purification techniques. As a result, attempts at either manipulating single adult-derived precursors or enriching them for therapeutic replacement have been difficult. The few reported successes at harvesting these cells from dissociates of adult brain, whether using avian (Goldman et al., 1992; 1996), murine (Reynolds and Weiss, 1992), or human (Kirschenbaum et al., 1994) tissue, have all reported less than 1% cell survival. Thus, several groups have taken the approach of raising lines derived from single isolated precursors, continuously exposed to mitogens in serum-free suspension culture (Reynolds and Weiss, 1992; Morshead et al., 1994; Palmer et al., 1995). As a result, however, many of the basic studies of differentiation and growth control in the neural precursor population have been based upon small numbers of founder cells, passaged greatly over prolonged periods of time, under constant mitogenic stimulation. The phenotypic potential, transformation state, and karyotype of these cells are all uncertain; after repetitive passage, it is unclear whether such precursor lines remain biologically representative of their parental precursors, or instead become transformants with perturbed growth and lineage control.
In order to devise a more efficient means of isolating native, unpassaged and untransformed progenitor cells from brain tissue, a strategy by which brain cells could be freely dissociated from brain tissue, then transduced in vitro with plasmid DNA bearing a fluorescent reporter gene under the control of neural progenitor cell-type specific promoters was developed (Wang et al., 1998). This permitted isolation of the elusive neuronal progenitor cell of the CNS, using the Tα1 tubulin promoter, a regulatory sequence expressed only in neuronal progenitor cells and young neurons.
Neuronal progenitor cells in the ventricular lining and dentate gyrus of the adult mammalian hippocampus are integral to learning, and to the acquisition and storage of new memories. Most dementing illnesses, including Alzheimer's and Parkinson's disease, involve the loss of either these cells or other hippocampal cells to which they connect; in addition, many epileptic syndromes involve the loss of these cells, including epilepsies that arise from brain trauma, birth injury, hypoxic injury, and some infections. Thus, a wide variety of neurological diseases share as a common feature damage to, or loss of, the hippocampal dentate cell population.
However, hippocampal progenitor cells have never been isolated. The existence of these hippocampal progenitor cells has been reported in adult animals ranging from chickadees to humans (Altman et al., 1965; Kaplan et al., 1977; Bayer et al., 1982; Barnea et al., 1994; Gould et al., 1997; Gould et al., 1998; Eriksson et al., 1998). In rodents, hippocampal neurogenesis can be modulated by stress (Gould et al., 1992), enrichment (Kempermann et al., 1997), exercise (van Praag et al., 1999), and learning (Gould et al., 1999). Among primates, both adult macaques (Gould et al., 1998; Kornack et al., 1999) and humans (Eriksson et al., 1998) exhibit histological evidence of neurogenesis in the dentate gyrus. Hippocampal cells have been found in suspension cultures derived from both adult rats (Palmer et al., 1997) and humans (Kukekov et al., 1999); these can expand in response to FGF2, include multipotential founders (Palmer et al., 1997), and are capable of heterotopic integration into other regions of granular neurogenesis, such as the olfactory subependyma (Suhonen et al., 1996). Yet despite the widespread incidence of hippocampal neurogenesis in adult animals, native hippocampal progenitor cells have never been separated and purified as such, in rodents or humans. As a result, no assessment of the abundance, factor-responsiveness, or regenerative capacity of these cells has been possible.
A strong need therefore exists for a new strategy for identifying, separating, isolating, and purifying native neural progenitor cells from hippocampal tissue.